Aptamers are single-stranded nucleic acid (DNA or RNA) ligands that possess a number of features that render them useful as therapeutic agents. They are relatively small (8 kDa to 15 kDa) synthetic compounds that possess high affinity and specificity for their target molecules (equilibrium dissociation constants ranging from, for example, 0.05-1000 nM). Thus, they embody the affinity properties of monoclonal antibodies and single chain antibodies (scFv's) with the chemical production properties of small peptides. While initial studies demonstrated the in vitro use of aptamers for studying protein function, more recent studies have demonstrated the utility of these compounds for studying in vivo protein function (Floege et al, Am J Pathol 154:169-179 (1999), Ostendorf et al, J Clin Invest 104:913-923 (1999), Dyke, Circulation 114(23):2490-7 (2006), Group, Retina 22(2):143-52 (2002), Group, Ophthalmology 110(5):979-86 (2003), Nimjee et al, Mol. Ther. 14(3):408-15 (2006), Nimjee et al, Trends Cardiovasc Med. 15(1):41-5 (2005), Nimjee et al, Annu. Rev. Med. 56:555-83 (2005), Rusconi et al, Nat. Biotechnol. 22(11):1423-8 (2004)). In addition, animal studies to date have shown that aptamers and compounds of similar composition are well tolerated, exhibit low or no immunogenicity, and are thus suitable for repeated administration as therapeutic compounds (Floege et al, Am J Pathol 154:169-179 (1999), Ostendorf et al, J Clin Invest 104:913-923 (1999), Griffin et al, Blood 81:3271-3276 (1993), Hicke et al, J Clin Invest 106:923-928 (2000), Dyke, Circulation 114(23):2490-7 (2006), Group, Retina 22(2):143-52 (2002), Group, Ophthalmology 110(5):979-86 (2003), Nimjee et al, Mol. Ther. 14(3):408-15 (2006), Nimjee et al, Trends Cardiovasc Med. 15(1):41-5 (2005), Nimjee et al, Annu. Rev. Med. 56:555-83 (2005), Rusconi et al, Nat. Biotechnol. 22(11):1423-8 (2004)).
As synthetic compounds, site specific modifications can be made to aptamers to rationally alter their bioavailability and mode of clearance. For example, it has been found that 2′fluoro pyrimidine-modified aptamers in the 10 kDa to 12 kDa size range have a short circulating half-life (˜10 minutes) following bolus intravenous administration but that simple chemical modification of the aptamer or conjugation of the aptamer to a high molecular weight inert carrier molecule (e.g., PEG) increases circulating half-life substantially (6-12 hours) (Willis et al, Bioconjug Chem 9:573-582 (1998), Tucker et al, J Chromatogr Biomed Sci Appl 732:203-212 (1999), Watson et al, Antisense Nucleic Acid Drug Dev 10:63-75 (2000)). Bioactive and nuclease resistant single-stranded nucleic acid ligands comprising L-nucleotides have been described (Williams et al, Proc. Natl. Acad. Sci. 94:11285 (1997); U.S. Pat. No. 5,780,221; Leva et al, Chem. Biol. 9:351 (2002)). These “L-aptamers” are reportedly stable under conditions in which aptamers comprising nucleotides of natural strandedness (D-nucleotides) (that is, “D-aptamers”) are subject to degradation.
Aptamers can be generated by in vitro screening of complex nucleic-acid based combinatorial shape libraries (>1014 shapes per library) employing a process termed SELEX (for Systematic Evolution of Ligands by EXponential Enrichment) (Tuerk et al, Science 249:505-10 (1990)). The SELEX process consists of iterative rounds of affinity purification and amplification of oligonucleotides from combinatorial libraries to yield high affinity and high specificity ligands. Combinatorial libraries employed in SELEX can be front-loaded with 2′modified RNA nucleotides (e.g., 2′fluoro-pyrimidines) such that the aptamers generated are highly resistant to nuclease-mediated degradation and amenable to immediate activity screening in cell culture or bodily fluids. (See also U.S. Pat. No. 5,670,637, U.S. Pat. No. 5,696,249, U.S. Pat. No. 5,843,653, U.S. Pat. No. 6,110,900, U.S. Pat. No. 5,686,242, U.S. Pat. No. 5,475,096, U.S. Pat. No. 5,270,163 and WO 91/19813.)
Over the past decade, the SELEX technology has enabled the generation of high affinity and high specificity antagonists to a myriad of proteins including reverse transcriptases, proteases, cell adhesion molecules, infectious viral particles and growth factors (see Gold et al, Annu Rev Biochem 64:763-97 (1995)). In particular, this technology has been employed to generate potent antagonists of coagulation factors, including factors VIIa, IXa, Xa and thrombin, transcription factors, autoimmune antibodies, cell surface receptors, as well as Von Willebrand factor and GPIIb-IIIa (see, for example, Rusconi et al, Thrombosis and Haemostasis 83:841-848 (2000), White et al, J. Clin Invest 106:929-34 (2000), Ishizaki et al, Nat Med 2:1386-1389 (1996), Lee et al, Nat Biotechnol 15:41-45 (1997), Nimjee et al, Annu. Rev. Med. 56:555-83 (2005)). (See also Published U.S. Application No. 20030083294 and documents cited therein, which documents are incorporated herein by reference as is Published U.S. Application No. 20030083294.)
It has been shown previously that the activity of aptamers can be reversed by using matched antidote oligonucleotides (Dyke, Circulation 114(23):2490-7 (2006), Rusconi et al, Nat Biotechnol. 22(11):1423-8 (2004), Rusconi et al, Nature 419(6902):90-4 (2002)); Published U.S. Application No. 20030083294). Joachimi et al (J. Am. Chem. Soc. 129:3036-3037 (2007)) have reported that a G-quadruplex-binding porphyrin can be used to control the anticoagulant activity of a G-quadruplex-containing aptamer (the porphyrin binding to guanine-rich motifs in the quadruplex).
The present invention results from the identification of agents (referred to below as “universal antidotes”) that can bind therapeutic or diagnostic nucleic acid molecules, such as aptamers, siRNAs, etc., in a sequence independent manner and modulate (e.g., inhibit or reverse) their activity. The universality of the antidotes disclosed herein translates into significant savings in time and cost from the standpoint of drug development. Further, the nature of these antidotes (detailed below) is such that the formation of double-stranded RNA helices is avoided and, therefore, the potential inflammatory response associated therewith.